Mechanisms of chromosomal aberration production I. Aberration induction by ultraviolet light

We have studied chromosomal aberration production in V-79 Chinese hamster tissue culture cells by UV light administered during the post-DNA-synthetic G₂ phase of the cell cycle. The treatment produced achromatic lesions and some chromatid deletions in the first post-irradiation mitosis, but no isochromatid deletions or chromatid exchange aberrations. In contrast, when G₂ UV-irradiated cells were examined in their second post-irradiation mitosis, there were significant yields of chromatid-type aberrations of all types, including isochromatid deletions and chromatid exchanges.

We have earlier reported²¹ that UV-irradiation during the pre-DNA-synthetic G₁ phase of the cell cycle induces only chromatid aberrations and also that most chromosomal aberration production by UV in G₁ can be photoreactivated in cells possessing the photoreactivating enzyme. We present here a model for chromosomal aberration production by UV. In the model all aberration production is enzymatically mediated, a consequence of the functioning of known molecular repair mechanisms. The important elements in the model are the following:

1. (1) The vertebrate chromosome is mononeme; i.e., contains but a single DNA double helix during the prereplication G₁ phase of the cell cycle.

2. (2) The UV-induced DNA lesion leading to the production of most aberrations is the cyclobutane dimer between adjacent pyrimidines in one polynucleotide strand.

3. (3) Single chain breaks appear at metaphase as achromatic lesions.

4. (4) Dimer removal sometimes leaves unrepaired single chain gaps, possibly as a result of incomplete excision repair.

5. (5) The single-stranded DNA opposite a single chain gap can be cleaved by a single-strand DNAase.

6. (6) Gaps are left in newly synthesized DNA polynucleotide chains opposite defective template chains (i.e., opposite dimers and chain breaks).

7. (7) Double-strand breaks present following local DNA replication may “spread” to the other chromatid by a recombinational process between template and new polynucleotide chains, one from each of the homologous double helices.

The model predicts the occurrence of isoachromatic lesions and of chromatid deletions paired (isolocus) with achromatic lesions. Though often not reported, both do, in fact, occur. In addition, the model accounts for the phenomenon of sister-chromatid exchange as a manifestation of a recombinational, or post-replication, repair mechanism. Finally, the model offers a simple interpretation of chromosomal aberration production by a variety of chemical agents.     

Studies on radiation-induced chromosome aberrations in mouse spermatocytes. I. Stage specificity and dose-response relationships of chromosome aberrations induced in mouse primary spermatocytes following X-irradiation

The cytological analysis of chromosome aberrations induced at diplotene, mid-pachytene, zygotene and leptotene stages following X-irradiation was performed at diakinesis-metaphase I in mouse spermatocytes. The dose-response relationships fitted well to linear equations for deletion-type aberrations at each stage, and to linear-quadratic equations for exchange-type aberrations at all stages except for leptotene. The radiosensitivity to chromosome aberration induction tended to increase gradually with progression through synaptic and post-synaptic stages, diplotene being the most sensitive. Chromatid exchanges were hardly observed at leptotene, the aberrations being mainly isochromatid fragments. On the contrary, chromatid exchanges and isochromatid deletions were mainly observed at later stages (zygotene-diplotene). The specificity of chromosome aberration induction in primary spermatocytes might be influenced by chromatin organization and chromosomal configuration peculiar to meiotic cells.     

The biological activity of hydrogen peroxide. I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts

The cytogenetic effect of hydrogen peroxide (H2O2) was investigated in human embryonic fibroblasts. Chromosome-type aberrations were found together with chromatid-type aberrations in metaphase cells harvested 24 h after a single 10-min treatment with 10(-5)-10(-3) M H2O2 in 0.9% NaCl solution. The chromosome-type aberrations were observed to be predominantly dicentrics and deletions. Both types of aberration showed a dose-response relationship to the dose of H2O2 over the range of 10(-5)-1.5 X 10(-4) M H2O2. The intercellular distribution of dicentrics showed a Poisson distribution. Centric and acentric rings and abnormal monocentrics were a minor fraction of the chromosome-type aberrations. The chromatid-type aberrations observed, such as breaks, exchanges and gaps, showed no dose-response relationship. The frequency of isochromatid breaks was higher than that of chromatid breaks and approximately 70% of the isochromatid breaks were found in the centromeric or pericentromeric region. The intercellular distribution of chromatid exchanges showed an over-dispersed distribution. The generation of aberrations by H2O2 was effectively suppressed by catalase and several scavengers of hydroxyl radicals (.OH) such as ethanol, dimethyl sulfoxide (DMSO) and mannitol. This result suggest that .OH plays an essential role in the generation of the chromosome aberrations by H2O2.     

Folic acid and chromosome breakage. III. Types and frequencies of spontaneous chromosome aberrations in proliferating lymphocytes

The types and frequencies of spontaneous chromosome aberrations were studied in human lymphocytes cultured for 96 h in minimal essential medium (MEM) or MEM without folic acid (MEM-FA). In both media, the most frequent aberrations were chromatid gaps, isochromatid gaps and chromatid breaks. Chromosome (isochromatid) breaks and dicentrics were seen less frequently. Neither of these less frequent aberrations was seen in 4000 cells from MEM, but both were seen in 4000 cells from MEM-FA.     

Inter- and intra-chromosomal distribution of chromatid breaks induced by X-rays during G2 in human lymphocytes

Cultures of blood from healthy adults were irradiated 48 h after stimulation with 240 R of X-rays and fixed after various time intervals (0–2 h, 2–4 h, 4–6 h). ³HTdR was added to several cultures after irradiation. Mitotic and labelling indices were used to distinguish between two cell samples inside the irradiated G₂ population: D − cells reaching mitosis without mitotic delay and a high frequency of chromatic breaks and D + cells with mitotic delay and which, during the delay, repair most of the damage produced. After R banding 450 chromatid deletions were located in each of the two cell samples. The D + cells showed a higher frequency of breaks than the D − cells with decreasing chromosome size, in the telomeric and centromeric region and in the junction between the R + and R − bands. These results can be interpreted as indicative of a non-random distribution of repair processes both between and within chromosomes.     

Radiation-induced chromatid aberrations in the bone marrow and lymph nodes of the rat during the first 24 h after irradiation

Wistar rats of both sexes were exposed to 100 R of X-rays. Chromatid-type aberrations in metaphase figures of bone marrow and lymph node cells were scored after 2, 4, 6, 8 and 24 h and 3, 5, 7, 9 and 24 h, respectively.The shape of the curve for chromatid plus isochromatid breaks in bone marrow cells versus time is exponential. It is suggested that this shape is mainly a consequence of the continuous entrance into mitosis of cells irradiated while in S phase, in addition to those that were irradiated in G₂. For lymph nodes the frequency of chromatid plus isochromatid deletions increased up to the 5th h, then began to fall off in a manner similar to that for the bone marrow. The difference in the shape of the two curves is the consequence of the different dependence on time for chromatid and isochromatid breaks in each tissue. While the frequency of chromatid breaks fell steeply with time both for the bone marrow and for lymph nodes, the frequency of isochromatid breaks remained nearly constant for bone marrow, whereas it rose to a peak at the 5th h for the lymph nodes.These differences are tentatively explained by a shift in the phases of the cell cycle sampled owing to the greater mitotic delay of G₂ cells in lymph nodes, with the suggestion that in the late S phase the frequency of isochromatid breaks is lower than in all other phases of the cell cycle.     

The UV sensitivity of the bromodeoxyuridine-substituted chromosomes in the Chinese hamster

Chinese hamster cells with chromosomes differently substituted for BUdR (TT-TT, TT-TB, TB-TB, TB-BB, where T is thymidine containing chromatid and B is BUdR substituted chromatid) were exposed to UV-light in phase G2 and chromosome aberrations (mainly chromatid breaks) were analysed. Breaks frequency per chromosome was proportional to BUdR content. No breaks were found in TT-TT chromosomes. The frequency of breaks per TB chromatid was similar with TT-TB and TB-BB chromosomes. In TB-BB chromosomes, however, virtually no breaks occurred in TB chromatids whereas in BB chromatids, their frequency was much higher than was expected.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.     

X-ray-induced chromosome aberrations in mouse dictyate oocytes. I. Time and dose relationships.

Structural chromosome aberrations were analyzed in superovulated metaphase-I oocytes of the mouse, Mus musculus, at various times after a single acute dose of 200 R of X-rays. The aberrations seen were of the chromatid type, i.e., chromatid interchanges, isochromatid deletions and chromatid deletions. The aberration frequency was low during the interval 24 h to 5 days between irradiation and ovulation; peak frequency was reached when irradiation was given 14 days prior to ovulation. A dose-response study was made 14 days prior to ovulation at doses of 50, 100, 200, 300 and 400 R. A curve of these data indicated that a significant two-track component was present for both interchanges and deletions. Centromere staining revealed that symmetrical and asymmetrical interchanges occurred at approximately equal frequency and also that the asymmetrical equivalent of crossing-over was induced at a measurable frequency.     

Formation of chromatid-type aberrations in G2 stage of the cell cycle

CHO cells were treated in G₁ stage of the cell cycle with chromosome-breaking agents that act in an S-dependent manner. The cells were challenged in G₂ stage, before fixation, with various inhibitors of DNA synthesis or repair. Short-wave UV, mitomycin C, decarbomyl mitomycin and 4-nitroquinoline oxide (4NQO) were used as chromosome-breaking agents. The inhibitors of DNA repair or synthesis used were hydroxyurea, aphidicolin and caffeine. Permeabilization of cells followed by a treatment with Neurospora endonuclease (a treatment to convert DNA single-strand breaks into double-strand breaks) did not have any influence on the frequencies of chromatid aberrations induced by the chemicals used, whereas with the inhibitors the extent of potentiation varied depending on the mutagen and the inhibitor used.     

Mechanism of formation of chromatid exchanges

Chinese hamster cells with different patterns of distribution of 5-bromodeoxyuridine (BrdUrd) between chromosome subunits were subjected, during the G2 stage, to UV irradiation, which only produced breaks in BrdUrd substituted DNA. The frequency of chromatid and subchromatid interchanges as well as isochromatid aberrations was estimated. It was found that only BrdUrd containing chromatids were involved into aberrations; this result challenges the so called "molecular theory" for aberration production proposed by Leenhouts and Chadwick. A very small increase of the aberration yield in chromosomes without BrdUrd may be connected with the action of UV on the frequency of recombination. The observed frequency of interchanges was not proportional to the BrdUrd content in chromosomes and depended on the time of its incorporation: more exchanges were induced in the chromatids incorporating BrdUrd during the last round of replication. These regularities may be connected with some molecular peculiarities of chromosome structure and function.     

The mutagenic activity of aflatoxin B1 in the Cricetulus griseus hamster and Macaca mulatta monkey

Chromosome aberrations were scored in bone marrow cells of Cricetulus griseus hamsters and Macaca mulatta monkeys given a single i.p. injection of aflatoxin B1 (AFB1). The mutagenic activity of AFB1 was assessed by the percentage of cells bearing aberrations and by the total frequency of chromosome and chromatid breaks. Chinese hamsters were treated with five different doses of AFB1 ranging from 1 microgram to 5 mg/kg (LD50/30 = 12.2 mg/kg) and the aberration yields at each AFB1 dose level tested were determined at 24 h intervals for 5 consecutive days. Compared to controls the increase in the two types of chromosome abnormalities was significant in all tests. At 5 mg/kg of AFB1 the tests were carried out over a period of 92 days to assure the analysis of aberration yields with time. All chromosome aberration assays conducted during this period showed significant increases in the frequencies of aberrant cells and chromosome and chromatid breaks in comparison to controls. Macaque monkeys were treated in the same fashion using 0.1 and 1.0 mg/kg of AFB1 and the dynamics of chromosome aberration yields was analyzed for a period of 730 days. Similarly as in the case of Chinese hamsters the percentage of cells with aberrations and the frequency of chromosome and chromatid breaks were always higher in this period than the control value. Long-term aberration yield data obtained experimentally were expressed in the form of analytical curves which allowed to establish the time when the yields of aberrant cells reached their maxima and when they returned to the control level. In both animal species tested the courses of analytical curves had a similar dynamics. Factors that might be responsible for a long-term persistence and relatively great fluctuations of the chromosome aberration yields encountered after a single injection of AFB1 are discussed in detail.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Analysis of structural and numerical chromosomal aberrations at the first and second mitosis after X irradiation of two-cell mouse embryos

Two-cell mouse embryos were X-irradiated in the late G2 phase in vivo. The first and second postradiation mitoses were analyzed for chromosomal anomalies. The majority of structural aberrations visible at the first mitosis after irradiation were chromatid breaks and chromatid gaps; only a few interchanges and dicentrics were observed. The aberration frequency resulted in a dose-effect relationship which was well described by a linear model. At the second mitosis 29% of the structural aberrations of the first mitosis were counted; the aberration quality changed only slightly. It is discussed whether these aberrations are to be considered "new," "derived," or unchanged transmitted aberrations. Contrary to the results obtained after irradiation of one-cell embryos, little chromosome loss was induced by radiation in two-cell embryos.     

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

The nature and repair of DNA lesions that lead to chromosomal aberrations induced by ionizing radiations

Short treatment (up to 1 h) of cytosine arabinoside (araC) increases the frequencies of aberrations induced by X-rays in human lymphocytes, evaluated at the first mitosis following stimulation, or as prematurely condensed chromosomes of G₀ nuclei. Parallel biochemical experiments using nucleoid sedimentation technique, demonstrate that araC inhibits rejoining of DNA-strand breaks effectively. These results point out that X-ray-induced short-lived DNA strand breaks lead to chromosomal aberrations in human lymphocytes.     

Types and frequencies of chromosomal aberrations induced by irradiation of different parts of interphase in Crepis capillaris

Quantitative and qualitative estimates of chromosomal damage in roots of Crepis capillaris were made in metaphase cells at many time intervals after irradiation with 200 or 400 rad of ⁶⁰Co gamma-rays. The results have confirmed the general pattern described for cells of other organisms, and have revealed in addition the following new facts. (1) The formation of aberrations of chromosome and chromatid type is not determined by the time of chromosome duplication alone. (2) The relative frequencies of different types of discontinuity form peaks with the following time succession: single gaps, chromatid breaks, isolocus breaks. (3) The location of peaks does not depend on the radiation dose, and shows no correlation which the time of synthesis. (4) Irradiation of G₂ induces a significant number of chromosome-type exchanges in Crepis. (5) Higher doses of radiation in G₂ favour the formation of chromatid over chromosome exchanges and of isochromatid breaks over chromosome breaks. A new interpretation of the production of certain types of aberration is discussed.     

The effect of wortmannin on radiation-induced chromosome aberration formation in the radioresistant tumor cell line WiDr

We analyzed the formation of radiation-induced chromosome aberrations in the cells of the radioresistant colon carcinoma cell line WiDr after treatment with wortmannin, an inhibitor of PI-3 kinases, including DNA-PK. Cells irradiated in G0/G1 phase with 200 kV X rays were treated with wortmannin before or after irradiation. Chromosome-type and chromatid-type aberrations were scored in metaphase cells by either Giemsa staining or FISH. Moreover, DNA-PK activity was measured in the absence and presence of wortmannin. In irradiated G0/G1-phase WiDr cells, only chromosome-type aberrations, including simple and complex exchanges and excess acentrics, were observed. After addition of 1 to 20 microM wortmannin, the formation of chromosome-type exchange aberrations was completely suppressed. The irradiated cells displayed exclusively chromatid-type aberrations including simple and complex chromatid exchanges and chromatid/isochromatid breaks. Whether the chromatid-type aberrations arise during G0/G1 as a result of homologous recombination processes coping with damaged DNA or whether DNA damage induced during G0/G1 phase persists until S and G2 phase and is then processed by homologous recombination pathways must be investigated further.     

Synaptonemal complex damage in relation to meiotic chromosome aberrations after exposure of male mice to cyclophosphamide

The genetic implications of induced synaptonemal complex (SC) damage are not known. However, on theoretical grounds, such aberrations could be involved in mechanisms leading to potentially heritable defects. Cyclophosphamide (CP), a chemical reported to cause structural and numerical chromosomal aberrations in the mouse, was used to determine if SC damage observed in meiotic prophase is related to subsequent metaphase chromosomal aberrations. Male mice were injected i.p. with CP. In some instances, mice were also injected simultaneously with tritiated thymidine to label DNA so that cells could be tracked autoradiographically through spermatogenesis. Prophase, primary metaphase (M1), and secondary metaphase (M2) samples were sequentially harvested at appropriate times from the same individual, and nuclei were examined for aberrations. Correlation coefficients between SC and metaphase chromosome aberrations were calculated. The inclusion of tritium labeling increased the number and significance of positive correlations. Positive correlations were found between (1) dose-dependent total SC damage and damage to M1, and to a lesser extent, M2 chromosomes; (2) SC breaks/fragments and M1 chains/rings as well as isochromatid breaks/fragments; (3) SC asynapsis and M1 chromatid breaks/fragments; (4) SC multi-axial configurations and M1 chains/rings as well as isochromatid and chromatid breaks/fragments; and (5) SC multi-axial configurations and M2 hyperploidy. These correlations do not define mechanistic or causal relationships between SC and chromosomal damage. However, taken together with the observation that induced SC damage is many times greater than ensuing metaphase chromosome damage, they substantiate SC analysis as a highly sensitive indicator of potentially heritable effects of this (and presumably other) genotoxic agents.     

Static Cytofluorometry and Fluorescence Morphology of Mitochondria and DNA in Proliferating Fibroblasts

The shape, distribution, and content of mitochondria in individual cells were examined during the cell cycle phases (G₀/G₁, S, G₂ mitosis) in living human fibroblasts by static cytofluorometry and fluorescence microscopy. The morphocytochemical evaluations were performed in cell cultures submitted to double supravital fluorochrome staining with Hoechst 33342 and DiOC₆ to label DNA and mitochondria, respectively. The staining modalities were based on the stability of mitochondrial labeling. The G₁ to early S phases were characterized by the presence of filamentous mitochondria, except during the early postmitotic period. During late S, G₂, and mitotic phases, mitochondrial mass reached its highest value and mitochondria became short and numerous. During the last stage of mitosis, mitochondria were distributed among daughter cells through a cytoplasmic bridge.